PROJECT SUMMARY/ABSTRACT Solid organ transplantation is currently the treatment of choice for children with a variety of end-stage organ diseases. The success of clinical transplantation is dependent on the use of potent immunosuppressive drugs to prevent rejection of the allograft. However, even with our arsenal of immunosuppressive agents, between 10-40% of pediatric transplant recipients will have a rejection episode in the first-year post-transplant. Clearly, acute rejection remains a major hurdle in pediatric solid organ transplantation and thus is the critical question to be addressed in the proposed mechanistic study. The CTOT-C ?Biomarkers for Post-Transplant Lymphoproliferative Disorders in Children? will enroll over 1000 pediatric recipients of liver, heart, kidney or intestinal grafts and thousands of individual blood samples will be collected, processed (whole blood, PBMC and plasma) and stored at Stanford. In this proposal, the unique CTOTC-06 cohort of samples, clinical data and established infrastructure will be utilized to identify novel and robust surrogate endpoints of allograft status. In preliminary studies we have identified microRNAs and cellular phenotypes that correlate with either acute rejection or stable graft function. We now propose to determine the exosome miRNome, analyze TCR and immunoglobulin heavy chain repertoires, and perform a multi-parameter analysis of the alloimmune response by mass cytometry with the goal of identifying biomarkers of stable graft function and acute rejection in recipients of liver, heart, kidney and intestine transplants. We hypothesize that we can identify unique cellular, molecular, and functional changes in the immune response that are associated with and predictive of graft outcomes. We will test this hypothesis in the following specific aims: Aim 1: Determine the impact of an allograft on the early post-transplant immune response. Aim 2: Establish novel diagnostic and predictive approaches to determine allograft status. The development of novel and robust surrogate endpoints of allograft status will be a major advance in the field of pediatric solid organ transplantation.